The research program of the Structural Immunobiology Unit (SIBU) is composed of two integrated themes: the study of large macromolecular signaling platforms known as the inflammasomes and the mechanisms of nucleic acid recognition by innate immune receptors. We use X-ray crystallography as the primary method to dissect the molecular details of innate receptors and signaling adapters, integrated with chemical tools that target these signaling pathways for potential therapeutic applications. Several families of the pattern-recognition receptors (PRRs) play important roles in the innate immune system, such as the Toll-like receptors (TLRs), the RIG-I-like receptors (RLRs), the NOD-like receptors (NLRs), the PYHIN family of receptors and the C-type lectin receptors (CLRs). A subset of the NLR and PYHIN family of receptors form large macromolecular signaling scaffolds known as the inflammasomes. Inflammasomes are large signaling platforms composed of the receptor (such as NLRP1, NLRP3, NLRP6, NLRP7, NLRC4/NAIP, AIM2 and IFI16), the adapter protein ASC, and the effector enzyme procaspase-1. Activation of the inflammasomes leads, at a minimum, to maturation of proinflammatory cytokines IL-1&#946; and IL-18, and pyroptosis. Blocking IL-1 activities using FDA approved drugs Anakinra, Rilonacept, and Canakinumab has shown phenomenal success in the treatment of several inflammatory disorders, a testament to the physiological importance of the inflammasomes. Because inflammasomes are implicated in a wide array of inflammatory processes in infectious diseases and autoimmunity, they are one of the cornerstones of the innate immune system. A major unresolved issue in the field has been the lack of definitive evidence for direct receptor:ligand association for most inflammasomes: seemingly unrelated stimuli are all capable of activating inflammasomes. Therefore, the true identities of the respective ligands and a unifying mechanism of inflammasome activation remain elusive. Nucleic acids are among the most potent activators of cells in the innate immune system that subsequently induce robust adaptive immunity. However, immune responses to nucleic acids, the universal genetic material, pose a unique challenge. By definition, PRRs from the host (self) typically recognize structural and chemical signatures that are specific for microorganisms (non-self). In contrast, innate DNA recognition is largely independent of sequences or modifications, and DNA from host, microbial, and synthetic sources are all known to induce inflammatory responses. It is now evident that the innate nucleic acid receptors play fundamental roles in anti-viral and anti-bacterial defense, as well as being an important contributor to autoimmune diseases such as lupus and psoriasis. During 2011-2012 we have focused on the following area of research. First, we studied the cytosolic DNA receptors AIM2 and IFI16 inflammasomes. The innate immune system responds to the presence of cytosolic DNA molecules through the secretion of interferons and proinflammatory cytokines as a host defense mechanism. The PYHIN family members AIM2 and IFI16 contain C-terminal DNA-binding HIN domain(s) and an N-terminal pyrin (PYD) domain that belongs to the death domain superfamily of signaling domains. AIM2 is predominantly a cytosolic protein that responds to dsDNA from both host and pathogens. The AIM2 inflammasome controls the activation of caspase-1 and subsequent maturation and secretion of IL-1&#946; and IL-18. IFI16 was originally identified as an anti-proliferative and pro-apoptotic nuclear protein. It was recently shown to be also present in the cytosol and was linked to interferon production in response to dsDNA. The precise signaling mechanisms downstream of IFI16 are less clear, although its ability to engage the ER resident protein STING (stimulator of interferon genes) appeared to be a prerequisite. Both AIM2 and IFI16 play crucial roles in host defense against intracellular bacteria such as Francisella tularensis and Listeria monocytogenes and DNA viruses such as vaccinia virus and HSV, as well as autoimmune diseases such as systemic lupus erythematosus (SLE) in which DNA is a major autoimmune target. We have determined the crystal structures of the AIM2 and IFI16 HIN domains in complex with dsDNA, and revealed the sequence-independent dsDNA recognition mediated by electrostatic attraction between the positively-charged HIN domain residues and DNA sugar-phosphate backbone. We illustrated an autoinhibited state of the AIM2 receptor in the absence of DNA-binding through intramolecular association of its AIM2 PYD and HIN domains. Lastly, we demonstrated that dsDNA can serve as both a ligand and an oligomerization platform for the formation of the AIM2 inflammasomes. Intriguingly, an antimicrobial peptide LL-37 that was shown to suppress AIM2 activation in a psoriasis model diminished AIM2-DNA association, suggesting a potential mechanism for therapeutic intervention of psoriasis using positively-charged peptides or small molecules. Complementary to our work on cytosolic DNA receptors, we investigated the roles of membrane bound protein RAGE in the induction of inflammation by nucleic acid. Endosomal DNA receptor such as TLR9 has limited access to extracellular nucleic acids, which is largely delivered by cell surface receptor RAGE and its co-receptor HMGB1 (high-mobility group protein B1). In collaboration with the Latz group at Germany, we found that RAGE enhanced immune response to DNA by promoting DNA uptake to the endosomal compartment, and RAGE-deficient mice can not mount typical lung inflammatory response to DNA. Fluorescence polarization, EMSA and confocal microscopy studies confirmed the direct binding of RAGE to DNA both in solution and on cell surface, as well as DNA-mediated oligomerization and clustering of RAGE. Interestingly, the ability of DNA to oligomerize RAGE appeared to be directly related to the kinetics of its trafficking through the endosomal network. In agreement, our recently determined crystal structure of the RAGE extracellular domain in complex with DNA demonstrated that DNA interacted with RAGE dimers through electrostatic attraction, similar to the DNA interaction with the HIN domains. In addition, the dimerization of the RAGE extracellular domains is mediated by a hydrophobic surface that may be targeted for the development of anti-inflammatory reagents, as the charge-based binding modality may be applicable for other RAGE ligands involved in inflammation, and diminishing the RAGE receptor association is expected to reduce the receptor:ligand association and signaling.